Protein Biosynthesis
translation
The Major Participants in
Translation
w mRNA
w tRNA
w Aminoacyl-tRNA synthetases
w Ribosomes
mRNA
The template for translation.
In prokaryotes, the mRNA is polycistronic
A polycistronic mRNA
AUG UAA AUG UAA AUG UAA
5’pppA/G
5’ UTR Cistron1 Cistron2 Cistron3 3’UTR
Monocistronic mRNAs in eukaryotes
Processing (CAPing, polyadenylation and
splicing ) of the primary transcripts usually
results in a a monocistronic mRNA.
A monocistronic mRNA
AUG UAA
Me7
Gppp (A)n=200
5’ UTR Open reading frame (ORF) 3’UTR
tRNA
The genetic code is read during translation via
adaptor molecules, tRNAs.
Aminoacyl-tRNA Synthetases
•Amino acids activation is to form aminoacyl-tRNAs
catalyzed by Aminoacyl-tRNA Synthetases
•This reaction activates the amino acid for protein
synthesis
Ribosomes
w Ribonucleoprotein particles
w Found in the cytosol, mitochondria and
chloroplasts
w Move along mRNAs, bind aminoacyl-tRNAs and
synthesize proteins
w Ribosomes are composed of 2 major units
Ribosome composition
Mitochondrial and chloroplast ribosomes differ from both
examples shown.
Ribosome Structure
w The two subunits assemble into complete
functional ribosome in the presence of mRNA.
w A tunnel runs through the large subunit.
w Growing peptide chain is thought to thread through
the tunnel during protein synthesis
A 3D model for the [Link] ribosome
30S
70S
50S
Comparison of
ribosomes and tRNAs
[Link] ribosome
tRNA binding sites:
the P site (peptidyl)
the A site (aminoacyl)
the E site (exit)
What are A, P and E sites on ribosome
• Sites in the interface between large and small ribosomal
subunits where tRNAs reside during protein synthesis
A-site is the position where the aminoacyl-tRNA binds
P-site is the position where peptide-tRNA binds
E-site is the spot where the uncharged tRNA is placed
• There are A, P and E sites on both subunits
movement of mRNA and tRNAs seems to be related to
a movement of the two subunits
• A- and P-sites have been known for long time, the E-site
was only discovered in the past decade after ribosome
structures have been solved
Process of protein synthesis
w Direction of peptide chain growth
N C
w polypeptides grow by addition of new
amino acid residues to the carboxyl end
Process of Prokaryotic Protein
Synthesis
w initiation
w elongation
w termination
Initiation
w Initiation involves binding of mRNA and
initiator aminoacyl-tRNA to a small subunit,
followed by binding of a large subunit
w Correct binding of mRNA on ribosome requires
alignment of a pyrimidine-rich sequence on 3'-
end of 16S RNA of 30S small subunit with a
purine-rich part of 5'-end of mRNA
w The purine-rich segment - the ribosome-binding
site - is known as the Shine-Dalgarno (S-D)
sequence
Shine-Dalgarno sequences recognized by [Link]
ribosomes
Initiation
w The initiator tRNA is one with a formylated
methionine: f-Met-tRNAfMet
w A formyl transferase adds the formyl group
from formyl-FH4.
w It is only used for initiation, and regular
Met-tRNAMet is used instead for Met addition
Events of Initiation
w 30S subunit with initiation factor proteins (IF-1 and
IF-3) binds mRNA
w IF-2 delivers the initiator tRNA in a GTP-
dependent process to the initiation codon.
w IFs, GTP, fMet- tRNAf, mRNA and 30S ribosome
form the 30S initiation complex
w Release of the IFs accompanying GTP hydrolysis
leads to binding of 50S subunit forming 70S
initiation complex.
When the two ribosomal subunits join, the
AUG initiator codon with its bound fMet-
tRNA aligns with the P site.
The P site is now occupied by initiator
fMet-tRNA, A site is empty.
w
initiation
30S subunit (IF-3:IF-
1) binds mRNA,
IF-2 delivers the
initiator f-Met-tRNA
to the initiation
codon
30S initiation complex
GTP hydrolysis is
accompanied by IFs
release and binding
of the 50S subunit
70S initiation complex
The Elongation Cycle
w Elongation: synthesis of peptide bonds -
with tRNAs bound to aminoacyl (A) and
peptidyl (P) sites.
w The elongation factors are vital to cell
function
Elongation factors
w EF-Tu Binds GTP,delivers aminoacyl-
tRNA to A site
w EF-Ts Displaces GDP from EF-Tu,
recycles EF-Tu-GTP
w EF-G Binds GTP, promotes
translocation of ribosome
Elongation step 1: Binding of an
aminoacyl-tRNA to A site
w Aminoacyl-tRNA binds to A site of ribosome
as a complex with EF-Tu and GTP
w GTP is then hydrolyzed and EF-Tu:GDP
complexes dissociate
w EF-Ts recycles EF-Tu by exchanging GTP for
GDP
Elongation second step:peptide
bond formation
w The amino N of the aa - tRNA in the A site
reacts with the carbonyl C of the aa (with
nascent chain) linked to tRNA in the P site.
tRNA P site tRNA A site
O O
Adenine O P O CH2 Adenine
O P O CH2 O
O
O − H H O− H H
H H H H
O OH O OH
O C O C
HC R HC R
NH :NH2
O C
HC R
NH3+
tRNA P site tRNA A site
O O
O P O CH2 Adenine O P O CH2 Adenine
O O
The nascent O − H H O − H H
H H H H
polypeptide, one OH OH O OH
O C
residue longer, is
HC R
now linked to the NH
tRNA in the A O C
site. HC R
NH
O C
HC R
NH3+
The tRNA in the P site is now unloaded (It will dissociate
during translocation.)
Peptidyl Transferase
w This is the central reaction of protein
synthesis
w 23S rRNA is the peptidyl transferase!
Ribosome is a ribozyme (catalytic rRNA)
w The "reaction center" of 23S rRNA is
located in the 50S particle - the catalytic
bases are among the most highly conserved
in all of biology.
Elongation step 3:translocation
w Ribosome moves one codon toward the 3’
end of the mRNA.
w Peptidyl-tRNA is translocated to P site from
A site.
w Uncharged tRNA is translocated to E (exit)
site, then dissociates from ribosome.
w Movements of the ribosome along mRNA
requires EF-G(translocase) and GTP.
Peptide Chain Termination
w Termination occurs when "stop codon"
reached
w Proteins known as "release factors"
recognize the stop codon at the A site
w Presence of release factors with a stop codon
at A site transforms the peptidyl transferase
into a hydrolase, which cleaves the peptidyl
chain from the tRNA carrier
Termination of protein synthesis
Players of translation
w Ribosome (RNA, proteins)
w mRNA
w tRNA
w Aminoacyl-tRNA synthetases
w Translation factors (initiation,
elongation,termination)
Events of Translation
w Initiation (different between prokaryotes and eukaryotes)
• Dissociation of ribosome
• Binding of initiation factor (IF1/eIF1A) to small particle
• Recruitment of f-Met-tRNAMet to small particle
• Recruitment of mRNA to small particle
• Binding of large particle - dissociation of initiation factors
w Elongation (similar between pro- and eukaryotes)
• Entry of aa-tRNA to A-site
• Peptide-bond formation
• Translocation of mRNA and tRNAs to P and E sites
• Entry of next aa-tRNA to A-site etc.
w Termination (similar between pro- and eukaryotes)
• Entry of release factor to recognize stop codon
• Exit of polypeptide and release factors
